腺相关病毒介导的shRNA敲低大鼠双侧臂旁外侧核EP3受体对发热反应的影响  

The effect of adeno-associated virus delivery of shRNA against EP3 receptors in the bilateral lateral parabrachial nucleus of rats on fever

作　　者：何田慧 王楠萍 吴思濠 魏滟麟 胥建辉[1] 张洁[1] He Tianhui;Wang Nanping;Wu Sihao;Wei Yanlin;Xu Jianhui;Zhang Jie(Key Laboratory of Thermoregulation and Inflammation of Sichuan Higher Education Institutes,Chengdu Medical College,Chengdu 610500;School of Clinical Medicine,Chengdu Medical College,Chengdu 610500)

机构地区：[1]成都医学院体温与炎症四川省高校重点实验室,成都610500 [2]成都医学院临床医学院,成都610500

出　　处：《安徽医科大学学报》2023年第11期1872-1877,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:32100926、31771289);成都医学院校基金(编号:CYTD19-04);成都医学院研究生创新基金(编号:YCX2022-01-01)。

摘　　要：目的研究腺相关病毒(AAV)介导的短发夹RNA(shRNA)敲低臂旁外侧核(LPB)EP3受体表达对LPB微量注射前列腺素E_(2)(PGE_(2))和腹腔注射脂多糖(LPS)引起的发热反应的影响。方法构建干扰EP3受体表达的AAV载体,采用立体定位方法分别向shRNA-control组和shRNA-EP3组大鼠LPB注射AAV2-CMV-EGFP和AAV2-shRNA-Ptger3(EP3)-EGFP病毒。4周后,利用荧光显微镜观察AAV病毒的转染效率;采用实时荧光定量PCR检测LPB EP3受体mRNA表达的变化,以验证敲低效果;采用无线遥测和能量代谢测定的方法检测LPB微量注射生理盐水、PGE_(2)或腹腔注射LPS对shRNA-control组和shRNA-EP3组大鼠的体温(T core)和能量代谢(EE)的影响。结果shRNA-EP3组及shRNA-control组大鼠AAV病毒转染主要集中于LPB脑区;与shRNA-control组相比,AAV病毒注射敲低了shRNA-EP3组大鼠LPB的EP3受体mRNA的表达(P<0.05);shRNA-control组和shRNA-EP3组大鼠的基础体温无明显差异,LPB微量注射生理盐水引起T core和EE均出现短暂轻微的升高,但2组间无明显差异;与shRNA-control组相比,PGE_(2)升高shRNA-EP3组大鼠T core作用明显减弱(P<0.05);shRNA-EP3组大鼠腹腔注射LPS后仍能引起发热反应,但与shRNA-control组相比,发热反应减弱,腹腔注射LPS后5.5 h的体温升高幅度明显降低(P<0.01)。结论敲低LPB的EP3受体表达减弱了LPB微量注射PGE_(2)和腹腔注射LPS引起的发热反应,提示,LPB的EP3受体介导LPB PGE_(2)的致热作用,部分参与了LPS性发热。Objective To investigate the effect of adeno-associated virus(AAV)delivery of short hairpin RNA(shRNA)against the Ptger3 gene in the lateral parabrachial nucleus(LPB)on the fever induced by microinjection of prostaglandin E_(2)(PGE_(2))into the LPB and the intraperitoneal injection of lipopolysaccharide(LPS).Methods AAV2-shRNA-Ptger3(EP3)-EGFP(shRNA-EP3)and AAV2-CMV-EGFP(shRNA-control)viruses were constructed and transfected the rat LPB by stereotaxic injection.Four weeks later,the transfection efficiency of AAV viruses was observed by fluorescence microscopy,and the knockdown efficiency was determined by real-time PCR of EP3 receptor mRNA on the LPB.The effects of microinjection of saline or PGE_(2)in the LPB or intraperitoneal injection of LPS on body temperature(T core)and energy expenditure(EE)of shRNA-control group and shRNA-EP3 group were monitored using an animal monitoring system with temperature telemetry.Results AAV virus transfection of shRNA-EP3 group and shRNA-control group was mainly found in the LPB.The EP3 receptor mRNA level in LPB of shRNA-EP3 group was reduced compared with that of shRNA-control group(P<0.05).There was no significant difference in basal body temperature between shRNA-control group and shRNA-EP3 group.T core and EE were briefly and slightly increased after microinjection of saline in the LPB,but there was no significant difference between the two groups.Compared with the shRNA-control group,the febrile response induced by LPB PGE_(2)was attenuated in the shRNA-EP3 group(P<0.05).Furthermore,the knockdown of EP3 receptor of LPB also attenuated the LPS-induced fever,and the T core 5.5 h post-LPS in the shRNA-EP3 rats increased compared with the baseline(P<0.05),which was lower than that in the shRNA-control rats(P<0.01).Conclusion EP3 receptor knockdown in LPB attenuates the febrile response induced by microinjection of PGE_(2)in the LPB and intraperitoneal injection of LPS,suggesting that EP3 receptors of LPB mediate the pyrogenic action of LPB PGE_(2)and partly participate in LPS

关 键 词：发热 EP3受体 臂旁外侧核 脂多糖 前列腺素E_(2) 

分 类 号：R338[医药卫生—人体生理学] R339.6[医药卫生—基础医学]